Alzheimer""s Disease (AD) is a neurodegenerative disorder characterized by a progressive decline in cognitive function, as well as by numerous amyloid plaques, neurofibrillary tangles (NFTs) and extensive neuronal loss in the brains of AD patients (Morrison-Bogorad et al., 1997, In The Molecular and Genetic Basis of Neurological Disease, Second edition, Butterworth-Heinemann, eds., pp 581-600). Although epidemiolgic studies have failed to identify a single cause of AD, genetic studies have implicated several mutations in three separate genes on different chromosomes that encode the amyloid-(A) precursor proteins (APP), presenilin-1 (PS-1), and presenilin-2 (PS-2) as the cause of autosomal dominantly inherited AD in a subset of kindreds with familial AD (FAD) (Van Duijn, 1996, J. Neurol. Neurosurg. Psychiatry 60:478-488; Goedert et al., 1997, In: The Molecular and Genetic Basis of Neurological Disease, Second edition, Butterworth-Heinemann, eds. pp. 613-628; Selkoe, 1997, Science 275:630-631). In addition, the 4 allele of the apolipoprotein E (APOE) gene has been shown to be a genetic risk factor for AD (Selkoe, 1997, Science 275:630-631). However, all of the known FAD mutations account for less than 5% of affected patients, since the majority of AD cases are sporadic and there is only modest evidence in support of familial clustering (Hardy, 1997, Proc. Natl. Acad. Sci USA 94:2095-2097).
Despite this heterogeneity, common factors may be involved in the pathogenesis of both hereditary and sporadic AD. These factors may promote the formation of A deposits and NFTs, as well as the massive degeneration of neurons in selected regions of all AD brains (Morrison-Bogorad et al., 1997 In: The Molecular and Genetic Basis of Neurological Disease, Second edition, Butterworth-Heinemann, eds. pp. 581-600). It has been suggested that the abnormal processing or production of A and plaque formation are pivotal events in the pathogenesis of the disease (Scheuner et al., 1996, Nature Med. 2:864-870; Mattson et al., 1992, Neurosc. 12:376-389). Furthermore, aggregated, but not monomeric species of A are hypothesized to induce the dysfunction and death of neurons in vitro by a range of mechanisms (Busciglio et al., 1995, Neuron. 14:879-888; Thomas et al., 1996, Nature 380:168-171; Behl et al., 1994, Cell 77:817-827). It has been hypothesized that AD brain regions which have accumulations of numerous A-rich senile plaques (SPs) are loci of elevated oxidative stress, perhaps reflective of an inflammatory reaction (Hensley et al, 1994, Proc. Natl. Acad. Sci. USA 91:3270-3274). Furthermore, it has been suggested that oxidant stress may be of functional importance in the pathogenesis of AD and that the production of reactive oxygen species (ROS) in the brain leads to lipid peroxidation and neuronal degeneration in AD (Gotz et al., 1994, Proc. Natl. Acad. Sci. USA 91:3270-3274).
Although there has been much speculation that ROS may play an important role in AD, there have been few data in support of this hypothesis. Efforts to elucidate the role of lipid peroxidation and oxidant stress in vivo have been hampered by the paucity of reliable quantitative molecular markers. Currently available molecular markers have been of limited value due to their chemical instability or their lack of sensitivity or specificity (Gutteridge and Halliwell, 1990, Trends Biochem. Sci. 15:129-1365).
The few studies which have been reported thus far of lipid peroxidation in the AD brain have provided evidence for increased lipid peroxidation by measuring levels of thiobarbituric acid reactive substances (TBARS) (Subbarao et al., 1990, J. Neurochem. 55:342-345; Palmer and Burns, 1994, Brain Res. 645:338-342; Lovell et al., 1995, Neurology 45:1594-1601; Balazs and Leon, 1994, Neuroch. Res. 19:1131-1137). However, the validity of this method is limited because it measures other aldehydes conjugated to TBARS, as well as non-lipid related chromogens. Recently, two separate groups of investigators have reported no difference in the level of TBARS and lipid hydroperoxides in AD versus control brains (Lyras et al., 1997, J. Neurochem. 68:2061-2069; Hayn et al., 1996, Life Sci. 59:537-544). Immunohistochemical data suggest the presence in AD brain of stable by-products of lipid peroxidation (Montine et al., 1997, J. Neuropath. Exper. Neurol. 56:866-871; Sayre et al., 1997, J. Neurochem. 68:2092-2097). While increased levels of 4-hydroxynonenal in post-mortem CSF of AD patient has been reported, no such quantitative data are available for this compound in AD brains (Lovell et al., 1997, Neurobiol. Aging 18:457-461).
Thus, there is an unmet need in the art for compositions and methods relating to molecular markers of oxidant stress or lipid peroxidation in a mammal for use in the diagnosis, treatment and development of therapeutics for diseases which manifest oxidant stress, such as Alzheimer""s disease. The present invention meets these needs.
The invention relates to a method of measuring the level of lipid peroxidation in a mammal suspected of having an oxidant stress syndrome or disease. The method comprises a) obtaining a first sample of a tissue or body fluid from the mammal; b) assessing the level of an isoprostane molecular marker for lipid peroxidation present in the first sample; and, c) comparing the level of the isoprostane molecular marker present in the first sample with the level of the isoprostane molecular marker present in a second sample of a tissue or body fluid obtained from an otherwise identical mammal which is not afflicted with an oxidant stress syndrome or disease, wherein an elevated level of the isoprostane molecular marker in the first sample relative to the level of the isoprostane molecular marker in the second sample, is indicative of an elevated level of lipid peroxidation in the mammal, thereby indicating the presence of an oxidant stress syndrome or disease in the mammal.
In one aspect, the method further comprises after a) and prior to b) isolating from the first sample the isoprostane molecular marker.
In another aspect, the elevated level of lipid peroxidation comprises an elevated level of a reactive oxygen species (ROS).
In yet another aspect, the elevated level of lipid peroxidation comprises an elevated level of inflammation.
In one embodiment, the elevated level of inflammation comprises elevated cyclooxygenase (COX) activity.
In yet a further aspect, the oxidant stress disease is Alzheimer""s disease.
In another aspect, the isoprostane molecular marker is selected from the group consisting of iPF2xcex1-III, iPF2xcex1-VI and 8,12-iso-iPF2xcex1-VI.
In an additional aspect, the tissue is brain tissue.
In one embodiment, the brain tissue is selected from the group consisting of brain frontal pole tissue and brain temporal pole tissue.
In another embodiment, the body fluid is selected from the group consisting of cerebrospinal fluid (CSF), plasma and urine.
The invention also relates to a method of diagnosing an oxidant stress syndrome or disease in a mammal. The method comprises a) obtaining a first sample of a tissue or body fluid from the mammal; b) assessing the level of the isoprostane molecular marker present in the first sample; and, c) comparing the level of the isoprostane molecular marker present in the first sample with the level of the isoprostane molecular marker present in a second sample of a tissue or body fluid obtained from an otherwise identical mammal which is not afflicted with the oxidant stress syndrome or disease, wherein an elevated level of the isoprostane molecular marker in the first sample relative to the level of the isoprostane molecular marker in the second sample, is indicative of an elevated level of lipid peroxidation in the mammal, whereby the oxidant stress syndrome or disease is diagnosed in the mammal.
In one aspect, the method further comprises after a) and before b) isolating from the first sample the isoprostane molecular marker.
Also included in the invention is a method of measuring the level of an isoprostane molecular marker for lipid peroxidation in a mammal suspected of having an oxidant stress syndrome or disease. The method comprises a) obtaining a sample of a tissue or body fluid from the mammal; b) isolating from the sample the isoprostane molecular marker by using a total lipids solvent extraction method; c) assaying the isoprostane molecular marker from b); and, d) quantifying the level of the isoprostane molecular marker.
In one aspect, the assaying comprises using a gas chromatography/mass spectrometry assay method which comprises a synthetic homologous isoprostane standard, and further wherein the quantifying is performed using peak area or peak height ratios.
In another aspect, the oxidant stress disease is Alzheimer""s disease. In yet another aspect, the isoprostane molecular marker is selected from the group consisting of iPF2xcex1-III, iPF2xcex1-VI and 8,12-iso-iPF2xcex1-VI.
In an additional aspect, the tissue is brain tissue.
In one embodiment, the brain tissue is selected from the group consisting of brain frontal pole tissue and brain temporal pole tissue.
In another aspect, the body fluid is selected from the group consisting of cerebrospinal fluid (CSF), plasma and urine.
The invention further relates to a method of identifying a compound useful for the treatment of Alzheimer""s disease in a mammal. The method comprises a) measuring the level of an isoprostane molecular marker for lipid peroxidation in either a sample of a tissue or body fluid obtained from a first mammal prior to administering the compound, or, in a sample of a tissue or body fluid obtained from an otherwise identical second mammal which is not to be administered the compound; b) administering the compound to the first mammal; c) thereafter measuring the level of the isoprostane molecular marker in a tissue or body fluid obtained from the first mammal; and, d) comparing the level of the isoprostane molecular marker measured in c) with the level of the isoprostane molecular marker measured in a), wherein when the level of the isoprostane molecular marker measured in c) is reduced relative to the level of the isoprostane molecular marker measured in a), a compound useful for the treatment of Alzheimer""s disease in a mammal is identified.
In one aspect, the isoprostane molecular marker of lipid peroxidation is selected from the group consisting of iPF2xcex1-II, iPF2xcex1-VI and 8,12-iso-iPF2xcex1-VI.
In another aspect, the tissue is brain tissue selected from the group consisting of brain frontal pole tissue and brain temporal pole tissue.
In another aspect, the body fluid is selected from the group consisting of cerebrospinal fluid (CSF), plasma and urine.
The invention also relates to a method of identifying an effective amount of a compound useful for the treatment of Alzheimer""s disease in a mammal. The method comprises a) measuring the level of an isoprostane molecular marker for lipid peroxidation in either a sample of a tissue or body fluid obtained from a first mammal prior to administering the compound, or, in a sample of a tissue or body fluid obtained from an otherwise identical second mammal which is not to be administered the compound; b) administering to the first mammal an amount of the compound; c) thereafter measuring the level of the isoprostane molecular marker in a tissue or body fluid obtained from the first mammal; and, d) comparing the level of the isoprostane molecular marker measured in c) with the level of the isoprostane molecular marker measured in a), wherein when the level of the isoprostane molecular marker measured in c) is reduced relative to the level of the isoprostane molecular marker measured in a), an effective amount of a compound useful for the treatment of Alzheimer""s disease in a mammal is identified.
The invention also includes a method of determining the optimal concentration of a compound useful for the treatment of Alzheimer""s disease. The method comprises monitoring the level of an isoprostane molecular marker for lipid peroxidation in a series of mammals administered the compound at a series of concentrations of compound, wherein the concentration of the compound which results in maximal reduction of the level of the isoprostane molecular marker in one or more of the series of mammals, which concentration is not toxic to the mammals, is the optimal concentration.
Also included is a method of determining the optimal dosage frequency of a compound useful for the treatment of Alzheimer""s disease. The method comprising monitoring the level of an isoprostane molecular marker for lipid peroxidation in a series of mammals administered the compound at a series of dosage frequencies, wherein the dosage frequency of the compound which results in maximal reduction of the level of the isoprostane molecular marker in one or more of the series of mammals, which dosage is not toxic to the mammals, is the optimal dosage frequency.
In one aspect, the compound is an antioxidant compound.
In another aspect, the compound is an anti-inflammatory compound, wherein the compound is administered at a series of concentrations effective to inhibit the activity of a cyclooxygenase (COX) enzyme in a mammal.
The invention further relates to a method of identifying a compound useful for reducing the level of an isoprostane molecular marker for lipid peroxidation in a sample of a tissue or body fluid obtained from a first mammal. The method comprises a) measuring the level of the isoprostane molecular marker in either a sample of a tissue or body fluid obtained from the first mammal prior to administering the compound, or, in a sample of a tissue or body fluid obtained from an otherwise identical second mammal which is not to be administered the compound; b) administering the compound to the first mammal; c) thereafter measuring the level of the isoprostane molecular marker in a tissue or body fluid sample obtained from the first mammal; d) comparing the level of the isoprostane molecular marker measured in c) with the level of the isoprostane molecular marker measured in a), wherein when the level of the isoprostane molecular marker measured in c) is reduced relative to the level of the isoprostane molecular marker measured in a), a compound useful for reducing the level of an isoprostane molecular marker in a mammal is identified.
In one aspect, the compound is present in an amount effective to inhibit the activity of a cyclooxygenase enzyme in the brain tissue of the mammal.
In another aspect, the compound is present in an amount effective to reduce the level of a reactive oxygen species in the brain tissue of the mammal.
In yet another aspect, the isoprostane molecular marker of lipid peroxidation is selected from the group consisting Of iPF2xcex1-III, iPF2xcex1-VI and 8,12-iso-iPF2xcex1-VI.
Also included is a kit for diagnosing Alzheimer""s disease in a mammal. The kit comprises a) a sample container for carrying a tissue or body fluid sample from the mammal; b) a solution for use in extraction of an isoprostane molecular marker for lipid peroxidation from the tissue or body fluid sample obtained from the mammal; c) a negative control solution of the isoprostane molecular marker of lipid peroxidation present at a concentration of about the concentration of the isoprostane molecular marker present in a tissue or body fluid sample of a mammal which is not afflicted with Alzheimer""s disease; d) a positive control solution of the isoprostane molecular marker of lipid peroxidation present at a concentration of about the concentration of the isoprostane molecular marker in a tissue or body fluid sample of a mammal which is afflicted with Alzheimer""s disease; e) an antibody directed against an isoprostane molecular marker for lipid peroxidation; and, f) an instructional material.
Further included is a kit for measuring the level of an isoprostane molecular marker for lipid peroxidation in a tissue or body fluid sample obtained from a mammal. The kit comprises a) a sample container for carrying a tissue or body fluid sample from the mammal; b) a solution for use in extraction of an isoprostane molecular marker of lipid peroxidation from the tissue or body fluid sample obtained from the mammal; c) a negative control solution of the isoprostane molecular marker of lipid peroxidation present at a concentration of about the concentration of the isoprostane molecular marker present in a tissue or body fluid sample of a mammal which is not afflicted with Alzheimer""s disease; d) a positive control solution of the isoprostane molecular marker of lipid peroxidation present at a concentration of about the concentration of the isoprostane molecular marker in a tissue or body fluid sample of a mammal which is afflicted with Alzheimer""s disease; e) an antibody directed against an isoprostane molecular marker for lipid peroxidation; and, f) an instructional material.